Noncyclic Notch Activity in the Presomitic Mesoderm Demonstrates Uncoupling of Somite Compartmentalization and Boundary Formation
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Downloaded from genesdev.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press RESEARCH COMMUNICATION in mice suggested both that FGF signaling acts upstream Noncyclic Notch activity of Notch and Wnt (Wahl et al. 2007) and that Wnt is in the presomitic mesoderm upstream of Notch and FGF (Dunty et al. 2008). Thus, it is still unclear how the activities of the Notch, FGF, and demonstrates uncoupling Wnt pathways are coordinated, and how these pathways of somite compartmentalization contribute to segment border formation and somite pat- terning in mouse embryos. and boundary formation Thus far, the most comprehensively studied genes dis- playing cyclic expression encode components of the Juliane Feller, Andre Schneider,1 Notch pathway. In mice, these are lunatic fringe (Lfng) Karin Schuster-Gossler, and Achim Gossler2 and the bHLH genes Hes1, Hes7, and Hey2 (McGrew et al. 1998; Jouve et al. 2000; Leimeister et al. 2000; Bessho Institute for Molecular Biology, Medizinische Hochschule et al. 2001). Notch1 activity itself oscillates in the pos- Hannover, D-30625 Hanover, Germany terior PSM and appears to be arrested in the anterior PSM through Mesp2-induced repression by Lfng (Morimoto et To test the significance of cyclic Notch activity for so- al. 2005), a glycosyltransferase that modulates the recep- mite formation in mice, we analyzed embryos expressing tiveness of Notch to various ligands (Hicks et al. 2000). activated Notch (NICD) throughout the presomitic me- Lfng expression is regulated by Notch activity (Morales soderm (PSM). Embryos expressing NICD formed up to et al. 2002), and Lfng was proposed to negatively regulate 18 somites. Expression in the PSM of Hes7, Lfng, and Notch1 activity in the PSM and to establish a negative Spry2 was no longer cyclic, whereas Axin2 was ex- feedback loop that drives cyclic Notch activity (Dale et pressed dynamically. NICD expression led to caudaliza- al. 2003; Morimoto et al. 2005). Similarly, Hes7 oscilla- tions are generated by a negative feedback loop of in- tion of somites, and loss of Notch activity to their ros- stable Hes7 protein periodically repressing Hes7 tran- tralization. Thus, segmentation and anterior–posterior scription (Bessho et al. 2001, 2003; Hirata et al. 2004). somite patterning can be uncoupled, differential Notch Mice homozygous for null alleles of Lfng and Hes7, re- signaling is not required to form segment borders, and spectively, display defects in somite compartmentaliza- Notch is unlikely to be the pacemaker of the segmenta- tion, and somites are irregular in form and size (Zhang tion clock. and Gridley 1998; Bessho et al. 2001). Studies in different vertebrate species addressing Supplemental material is available at http://www.genesdev.org. Notch function during somitogenesis have led to various Received March 20, 2008; revised version accepted June 17, views as to the role of Notch in this process. It has been 2008. proposed that the segmentation clock regulates the pe- riodic activation of Notch (Pourquie 1999; Serth et al. 2003), its signaling is required for the synchronization of Somitogenesis subdivides the paraxial mesoderm of ver- the clock in neighboring cells (Jiang et al. 2000; Ozbudak tebrate embryos into a series of homologous subunits, and Lewis 2008), and the Notch pathway is part of the the somites. Somites form sequentially at the anterior oscillator (Holley et al. 2002; Morales et al. 2002; Dale et end of the presomitic mesoderm (PSM). A molecular os- al. 2003; Morimoto et al. 2005; Mara et al. 2007). In cillator referred to as “segmentation clock” directs cy- mouse embryos, extensive genetic studies led to the clic expression of genes in the PSM and is coupled with view that in the anterior PSM, Mesp2-induced down- the progression of somitogenesis. Expression of cyclic regulation of Notch activity leads to somite boundaries genes is coordinated such that one wave of expression where cells with activated Notch1 abut cells with re- passes through the PSM during the formation of one so- pressed Notch1 activity, that the generation of somite mite (Palmeirim et al. 1997; McGrew et al. 1998; Jiang et polarity and borders is coupled, and Notch is part of the al. 2000; Jouve et al. 2000; Aulehla et al. 2003). Microar- core of the segmentation clock (Takahashi et al. 2003; ray studies of the mouse PSM transcriptome showed in Morimoto et al. 2005; Saga 2007). This contrasts with part mutually exclusive activation of the Notch, FGF, recent findings in zebrafish that indicated a role for and Wnt pathways during each cycle, suggesting coordi- Notch signaling merely in the posterior PSM in the syn- nated regulation of these three pathways (Dequeant et al. chronization of cells (Ozbudak and Lewis 2008). To fur- 2006). There is evidence that Wnt activity in the PSM ther study the role of Notch activity in somite formation acts upstream of Notch (Aulehla et al. 2003; Hofmann et and patterning in mice, we analyzed embryos expressing al. 2004). However, cyclic Lfng expression was main- activated Notch (NICD) throughout the PSM. Our re- tained in embryos with constitutive Wnt activity in the sults argue against a role for Notch as a pacemaker of the PSM (Aulehla et al. 2007). Similarly, the epistatic rela- clock, show that the confrontation of domains with and tionship of FGF and Wnt signaling is not clear, as studies without Notch activity is not a prerequisite for border formation, and demonstrate that segmentation and an- terior–posterior somite patterning can be uncoupled. [Keywords: Notch signaling; segmentation; somite patterning; somito- genesis] Results and Discussion 1Present address: Max-Planck-Institut für Herz- und Lungenforschung, D-61231 Bad Nauheim, Germany To activate Notch signaling throughout the PSM, we 2Corresponding author. E-MAIL [email protected]; FAX 49-511-532-4283. generated transgenic mice expressing Cre in the primi- Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.480408. tive streak under the control of regulatory elements of 2166 GENES & DEVELOPMENT 22:2166–2171 © 2008 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/08; www.genesdev.org Downloaded from genesdev.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press Notch in boundary formation with clearly discernable segment borders (Fig. 1G–K), in- dicating that the presence of activated transgenic Notch (exoNICD) throughout the PSM did not prevent border formation. To address how activation of endogenous Notch1 is affected by exoNICD, we analyzed the presence of en- dogenous NICD using an antibody that specifically rec- ognizes the cleaved form of Notch1 (but not the trans- gene-encoded NICD that lacks the V1744 epitope). In wild-type embryos, activated Notch1 (for clarity, from hereon referred to as endoNICD) was found in the pos- terior PSM in variable patterns, and in one or two bands in the anterior PSM (red lines in Fig. 2A–C), reflecting cyclic Notch activity. In contrast, T-NICD embryos (n = 21) showed one narrow stripe of endoNICD at the anterior end of the PSM (red arrowheads in Fig. 2D,E) that resembled the anterior endoNICD stripe of wild- type embryos, although levels appeared lower. In addi- tion, a subset of T-NICD embryos (n = 13) had an addi- tional weaker, fuzzy endoNICD stripe posterior to the anterior stripe (Fig. 2D, white arrowhead). In the poste- rior PSM region of T-NICD embryos, endoNICD was severely down-regulated (Fig. 2D,E), indicating that acti- vation of Notch1 was disrupted in the T-NICD embryos. The presence of a stripe(s) of endoNICD in the anterior Figure 1. Segmentation in embryos with constitutive Notch activ- ity. (A–F) In situ hybridization of embryos showing expression of the Nicd-Gfp fusion transcript throughout the paraxial mesoderm of T(s)ϻCre; R-NICD embryos (A,B) and up-regulation of Hey1 in the PSM and the somites (C,D) compared with wild-type embryos (E,F). (G,J,L) Scanning electron microscopic pictures illustrating somites in T(s)ϻCre; R-NICD (G,J) and wild-type (L) embryos. (H,I,M,N) He- malaun-stained plastic sections of somites in T(s)ϻCre; R-NICD (H,I) and wild-type (M,N) embryos. (K) Segmented paraxial meso- derm in T(s)ϻCre; R-NICD embryos visualized by Myf5 expression. Developmental stages are indicated on top. Bars: H,I,M,N, 500 µm. the brachyury gene (Stott et al. 1993), and crossed these mice to ROSANICD mice that allow for conditional ex- pression of the constitutively active intracellular do- main of Notch1 (NICD) by Cre-mediated excision of a stop cassette (Murtaugh et al. 2003). Cre activity resulted in reporter gene expression throughout mesodermal tis- sues (Supplemental Fig. 1), and in embryos carrying the ROSANICD allele and the Cre transgene (hereafter re- ferred to as T-NICD embryos), NICD-Gfp fusion tran- scripts were detected throughout the PSM and in Figure 2. Endogenous Notch1 activity in wild-type and mutant somites (Fig. 1A,B). The Notch target Hey1 was strongly embryos. (A–H) Immunohistochemical detection of activated en- up-regulated throughout the paraxial mesoderm of dogenous Notch1 in the PSM. In contrast to wild-type embryos T-NICD embryos (Fig. 1, cf. C,D and E,F) demonstrating (A–C), endogenous Notch1 activity is down-regulated in the poste- activation of the Notch pathway. T-NICD embryos did rior but is found in one or two stripes in the anterior PSM (arrow- heads in D,E) of T(s)ϻCre; R-NICD embryos. (F) Specificity of anti- not complete turning, became severely retarded and dis- body is demonstrated by the lack of staining in T(s)ϻCre; N1loxp/loxP torted after embryonic day 9.5 (E9.5) with impaired elon- embryos. Loss of Dll1 (G)orPofut1 (H) function completely abol- gation of the embryonic axis, and died around E10.5 with ishes Notch1 activity.